Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
The widespread interest in the potential of adenoviruses as therapeutic vectors has overshadowed the utility of hybrid viruses as tools to study intracellular processes. The origins of such research began with the analysis of human adenovirus-SV40 hybrids that were capable of replication in otherwise nonpermissive monkey cells because of the expression of a domain of the SV40 T antigen [reviewed in Klessig (1984)], but the full potential of this approach was realized only when it became practicable to create the desired recombinant genomes in vitro [for early reviews see Gluzman et al. (1982) and Berkner (1988)]. Although adenovirus vectors expressing a wide variety of individual gene products have been produced, very few have been created to investigate the mechanisms underlying DNA repair or recombination. The exceptions include those that express the cre site-specific recombinase, which has been used to restructure viral and cellular genomes in vivo (Anton and Graham, 1995; Wang et al., 1995; Parks et al., 1996; Hardy et al)., 1997; Kanegae et al., 1995; Wang et al., 1996), and the bacteriophage T4 endonuclease denV, which was shown to be capable of functioning as a replacement for the mutant protein in xeroderma pigmentosum cells of groups A, C, and E (Colicos et al., 1991). These experimental precedents suggest that adenovirus vectors hold great promise as tools to investigate DNA metabolism. They can be used to infect a wide range of cell lines from a variety of species, including cells with deficiencies in DNA repair.
Among the processes that might be investigated using adenovirus vectors are those involved in double-strand break (DSE) repair (DSBR) in mammalian cells. The mechanisms by which mammalian cells repair DNA double-strand breaks (DSBs) are not fully understood at the molecular level. The nuclear-replicating DNA-containing viruses are not only subject to these repair mechanisms, but can also be used to investigate them. One way to exploit the virus systems is to construct vectors that express endonucleases that create DSBs in specific targets and then to follow DSB repair under a variety of experimental conditions. Double-strand breaks arise in cellular DNA as the result of the action of DNA-damaging agents and of normal cellular processes such as immunoglobulin gene rearrangement. Double-strand breaks interfere with cellular DNA replication and chromosome segregation and are lethal if unrepaired. Therefore, mammalian cells contain an efficient double-strand break repair (DSBR) system that rapidly joins free double-stranded ends by an homology-independent mechanism (Jeggo, 1998). The cellular DSBR system is active both on broken chromosomal DNA and on exogenous DNAs introduced into cells, and the linear adenovirus genome is a potential substrate for concatemerization or circularization by the DSBR system in infected cells. DSBR is not only crucial to the survival of the cell, but also is involved in the developmentally regulated rearrangement of B cell and T cell receptor loci [for a recent review see Lieber (1998)]. Genetic and biochemical data from both mammalian cells and fungi show that DSBR depends on the activities of a large set of proteins including DNA-dependent protein kinase (PK); DNA ligase IV and its stimulatory protein, the product of the XRCC4 gene; and the protein products of the Mre11, Rad50, and mammalian NBS or the yeast equivalent Xrs2 genes. Recently, an in vitro system for DSBR, which is dependent on this set of proteins (Baumann and West, 1998), was described. However, despite advances in identifying the components of DSBR in mammalian cells, many questions remain about the precise mechanisms operating on DSBs occurring in vivo, and there continues to be a need for simple assays for DSBR. Until recently, such assays have depended either on the transfection of restriction enzyme-cleaved substrate DNA [reviewed in Roth and Wilson (1988)] or on the introduction of purified restriction enzymes themselves into the cell [see for example Phillips and Morgan (1994); Liu and Bryant (1993); Costa and Bryant (1991)]. Subsequent analyses of the fates of the broken DNAs have usually taken place after a long period of time and/or the selection of specific recombinant products. Despite the success of these methods in suggesting several important aspects of DSBR in mammalian cells (Roth and Wilson, 1988), they do not allow the fate of DSBs created in vivo at specific genetic locations to be followed in real time. This approach has been particularly productive in Saccharomyces cerevisiae, in which the mating type switch HO endonuclease and its recognition site have been exploited to examine the mechanisms of both homologous and nonhomologous recombinational repair [reviewed in Haber (1995)]. Most recently, conditional expression of the EcoRI endonuclease has allowed detailed genetic analysis of those genes essential for nonhomologous DSBR in S. cerevisiae (Lewis et al., 1998, 1999). In mammalian cells, the development of cell lines and plasmid vectors expressing the intron-encoded enzyme I-SceI has allowed a closer look at the repair of specific DSBs, created and resolved in vivo (Rouet et al., 1994a,b; Sargent et al., 1997; Choulika et al., 1995). This system has been used, for example, to show that mutations in Ku80 abolish end-joining, while permitting normal levels of homologous recombination (Liang et al., 1996), to demonstrate loss of heterozygosity following the repair of an induced DSB (Moynahan and Jasin, 1997), to estimate the lengths of gene conversion tracts (Elliott et al., 1998), and to examine the control of translocation (Richardson et al., 1998). This in vivo approach is more likely to give a true picture of the physiological consequences of the formation and processing of DSBs.
Adenovirus vectors expressing the yeast mating-type switching endonuclease HO may be used to examine the formation and fate of double-strand breaks created in adenovirus genomes containing an HO recognition site. The results show that the HO recognition site can be cleaved by the HO gene product in mammalian cells, but that in permissive infections repair is below the limits of detection of the methods employed. Broken genome fragments accumulate and those containing packaging signals can be encapsidated. However, in nonpermissive infections in which E4 product expression is absent or severely reduced, endjoining of fragments takes place, suggesting that one or more E4 products inhibit DSBR.
The cellular DSBR system is active both on broken chromosomal DNA and on exogenous DNAs introduced into cells, and the linear adenovirus genome is a potential substrate for concatemerization or circularization by the DSBR system in infected cells. However, in wild-type adenovirus infections intracellular adenovirus DNA, with the exception of branched replication intermediates, is almost exclusively monomeric and linear. Weiden and Ginsberg (1994) reported that adenovirus mutants lacking early region 4 (E4) produced concatemers of viral DNA in infected Hela cells, and that the presence of either E4 ORF6, which encodes a 34 kDa protein (E4 34k), or E4 ORF3, which encodes an 11 kDa protein (E4 11k), suppressed concatemer formation. End-to-end joining by the DSBR system was suggested as a mechanism for concatemer formation. If concatemers arise in that way, the E4 proteins might prevent concatemerization by antagonizing DSBR in infected cells. As disclosed herein, that hypothesis was investigated by examining concatemer formation in cells that lack the cellular DNA-dependent protein kinase (DNA PK), an essential element of the DSBR system (Jeggo, Taccioli, and Jackson, 1995), by assessing the effects of E4 products on DSBR-dependent V(D)J recombination, by assessing the effects of E4 products on DSBR-dependent V(D)J recombination, and by examining the effects of E4 products on repair by the DSBR system of double-strand breaks in the viral genome induced by a site-specific endonuclease, the yeast mating type switching endonuclease (HO) endonuclease.